Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Conjugation of polymers to peptide and protein therapeutics to generate hybrid molecules with unique and distinct molecular properties has become a popular approach to alter and/or control their stability, biodistribution, pharmacokinetics and toxicology. Since the pioneering work of Abuchowski et al. (1997a & b) of grafting polyethylene glycol (PEG) chains to albumin, PEGylation has been a very widely used conjugation approach to generate protein-polymer bio-conjugates of unique biological properties or diminished toxicities.
The first step of the PEGylation of proteins and peptides can involve functionalizing PEG with group specific reagents, so that the conjugation of PEG to protein can be targeted to specific side chain groups of the proteins, such as amino, carboxyl, sulfhydryl or guanidino groups. More recently, the strengths of site directed mutagenesis have also been integrated to achieve site specific PEGylation. Cysteine (Cys) residues can be introduced in a site specific fashion in place of preselected surface amino acid residues of proteins. The thiol groups of the newly introduced Cys residues can be targeted for PEGylation using maleimide chemistry based PEG reagents. Replacement of Serine (Ser) or Threonine (Thr) with Cys has an advantage that the net charge of the mutant protein is not altered as a result of the PEGylation, i.e. a conservative PEGylation protocol as far as the site directed mutagenesis of the parent protein is conservative.
A chemical approach to introduce new thiols on the ε-amino groups of proteins as a means of increasing accessibility of the surface amino groups for PEGylation and targeting the PEG reagents to these sites by maleimide chemistry has been developed (Acharya et al., 1996). The initial approach involved thiolation of amino groups of proteins using 2-iminothiolane. In a preferred protocol, protein is incubated with iminothiolane in the presence of PEG maleimide, and the new thiol groups generated in situ on the protein amino groups are trapped immediately by PEG maleimide as succinimidyl derivatives (Acharya et al., 2003).
Hemoglobin (Hb) based blood substitutes are being developed to overcome shortages of blood supply (Chang, 1999; Klein, 2000). The most extensively studied and financed blood substitute has been diaspirin cross-linked Hb. Though intramolecular crosslinking of Hb helped to overcome nephrotoxicity and high oxygen affinity of acellular Hb, the two major limitations of stroma-free Hb as a blood substitute (Chang, 1999), the product remained vasoactive (Alayash et al., 2001; Kramer, 2003; Winslow, 2000). The vasoactivity has been attributed to the extravasation of acellular Hb and the scavenging of nitric oxide (NO) by the extravasated Hb.
Enhancing the molecular size of Hb by oligomerization to prevent or reduce the extravasation of Hb has been one of the solutions advanced to overcome the vasoactivity of acellular Hb, while another is to lower the affinity of Hb to nitric oxide by site directed mutagenesis. Animal studies have shown that both approaches reduce the pressor effect of Hb (Gulati et al., 1999).
An alternate approach to overcome the vasoactivity of Hb involves engineering the properties of plasma volume expanders into Hb, namely high viscosity and high colloidal osmotic pressure to Hb (Acharya et al., 2005). Enzon PEGylated bovine Hb, which carries ten copies of PEG-5K chains, was found to be nonhypertensive even though its affinity for NO is comparable to that of other modified Hbs that are under clinical trial. The unusual molecular properties of Enzon PEGylated bovine Hb, namely enhanced molecular volume, high viscosity and high colloidal osmotic pressure, which are also the properties of plasma volume expanders, have been attributed as the molecular basis of the neutralization of vasoactivity of acellular Hb. Accordingly, PEGylation of Hb has been considered as an approach to generate non-hypertensive Hb. This application of polyethylene glycol (PEG), a water-soluble, inert and nontoxic polymer, reflects a very different translation of PEGylation than other applications of other molecular properties induced to proteins by PEGylation, where PEGylation-induced increased solubility, increased half-life, and reduced access of molecular surface to the immune system are used to generate PEG-protein conjugates of therapeutic value (Bailon and Berthold, 1998; Harris et al., 1997, 2003).
In an attempt to establish that the neutralization of vasoactivity is a generalized consequence of PEGylation induced molecular properties of acellular Hb and not unique to Enzon PEGylated bovine Hb, a hexaPEGylated human Hb [(SP-PEG-5K)6-Hb] was generated using a PEGylation platform referred to as extension arm facilitated PEGylation protocol (Acharya et al., 2005; Manjula et al., 2005). This hexaPEGylated Hb exhibited an unusually high increase in the molecular volume, increased viscosity and higher colloidal osmotic pressure and was non-hypertensive.
The non-hypertensive PEGylated human Hb, (SP-PEG5K)6-Hb, carries only six copies of PEG-5K chains while the Enzon PEGylated bovine Hb carries ten copies of PEG-5K chains. In addition, the chemistry of conjugation of PEG-chains in the two products is very distinct. In the decaPEGylated bovine Hb of Enzon, the PEG-chains are conjugated to the surface amino groups of Hb through an urethane linkage, which results in the loss of the positive charge of the {acute over (ε)}-amino group of Lysine (Lys) residues to which PEG chains are conjugated. In the hexaPEGylated human Hb, the PEG-5K chains are conjugated to the surface amino groups using the extension arm facilitated PEGylation. In this protocol, the surface amino groups are first reacted with iminothiolane, which results in the extension of the side chain of Lys residues by the linking of δ-mercapto butirimidyl chains, and the thiol groups of the extension arm are modified with maleimide PEG (Acharya et al., 2005; Manjula et al. 2005). The higher efficiency of the PEG-chains of hexaPEGylated Hb to neutralize the vasoactivity could be a correlate of the fact that the extension arm facilitated PEGylation conjugates the PEG-chains without changing the surface charge of Hb, i.e. the PEGylation is conservative. Since the colloidal osmotic pressure (COP) of the hexaPEGylated Hb is comparable to that of decaPEGylated Hb, the results reflect the role of the conservation of the positive charge of Hb at the sites of PEGylation. When the surface charge is conserved, the PEG-chains conjugated are possibly more efficient in inducing the desirable molecular properties to Hb that facilitates the neutralization of its vasoactivity.
To gain further insight into the possible advantages of conserving the surface charges at the site of the conjugation, another hexaPEGylated Hb has been generated in which the charge of the surface amino groups is conserved. {acute over (ω)}-methoxy PEG 5K-propoinaldehyde is conjugated to Hb in the presence of sodium cyanoborohydride (reductive alkylation chemistry). The molecular properties of this PEG-Hb conjugate, particularly the COP of (Propyl-PEG5K)6-Hb was considerably higher than that of [(SP-PEG-5K)6-Hb], leading to the suggestion that either the chemistry of conjugation of PEG-chains to Hb or the site selectivity of PEGylation of Hb influences the molecular properties of the PEG-Hb (Hu et al., 2005).